KR100830811B1 - Bio-sensor for detecting tumor marker - Google Patents

Abstract

The present invention provides a biosensor for detecting a tumor marker, which can easily and selectively detect tumor markers in real time. The present invention also provides a biosensor manufacturing method for detecting tumor markers. The present invention also provides a method for analyzing tumor markers using the biosensor of the present invention described above. Biosensor for detecting tumor markers provided by the present invention, the substrate; A first electrode attached to the substrate; A second electrode attached to the substrate; Semiconducting carbon nanotubes electrically connecting the first electrode and the second electrode; A linker molecule whose one end is bonded to the surface of the semiconducting carbon nanotube; And a bio-receptor for a tumor marker that is bound to the other end of the linker molecule.

Biosensor, tumor marker, carbon nanotube, electric field effect

Description

Biosensor for detecting tumor markers

1 is a cross-sectional view schematically showing one embodiment of the biosensor of the present invention.

Fig. 2 is an AFM (atomic force microscope) photograph of the transducer part element manufactured in the present example.

3 is an AFM photograph showing that the bioreceptor is immobilized on the surface of the carbon nanotube of the biosensor of Example 1. FIG.

4 is a graph showing current changes of four CEA samples measured in Example 2. FIG.

FIG. 5 is a graph showing current changes of three CEA samples measured in Example 3. FIG.

6 shows the correlation between the ratio (%) of the current reduction value (ΔI) to the initial current (I ₀ ) with no CEA sample solution dropping and the CEA concentration using the results shown in FIG. 5. The graph shown.

The present invention relates to a bio-sensor, and more particularly to a bio-sensor for detecting tumor marker.

Biosensors, for example, use biomaterials (bio-receptors) that can recognize specific chemicals (target bio-materials), such as enzymes, antigens, antibodies, etc. It is a device that quickly, easily and selectively measures chemicals.

The biosensor generally consists of a sensor part and a transducer part. The sensor unit consists of a bio-receptor that selectively binds to a target bio-material. The transducer unit outputs an electrical signal corresponding to the electrochemical change caused by the combination of the target bio-material and the bio-receptor. Representative types of biosensors include enzyme sensors and immune sensors.

Examples of enzyme sensors include enzyme electrode sensors, enzyme transistor sensors, enzyme thermistor sensors, and the like. The enzyme electrode sensor measures the membrane potential difference generated by the catalytically active function of the enzyme in the ion-selective sensitive membrane (potentiometric type), or measures the current value due to the reaction between the substance and the electrode (current Measurement type) to measure the concentration of a specific substance involved in the enzymatic reaction. The enzyme transistor sensor is one in which an enzyme causing a pH change is immobilized on the gate surface of an ion sensitive field effect transistor. Enzyme thermistor sensor combines the immobilized enzyme and thermistor which measures the heat of chemical reaction. Examples of practical enzyme sensors include glucose sensors, alcohol sensors, organic acid sensors, amino acid sensors, urea sensors, and the like that measure blood glucose levels.

Immune sensors are used to measure proteins, antigens, hormones, pharmaceuticals, etc., which are present in body fluids such as blood, for example, by using the selective binding force between antigens and antibodies. Representative examples of the immune sensor include an electrode type immune sensor. The electrode type immune sensor is composed of an antigen or antibody immobilization membrane and an electrode. When a substance such as a negatively charged protein binds to the surface of the antibody immobilized membrane that is positively charged, the positive charge of the antibody immobilized membrane is reduced. At this time, by measuring the potential change of the antibody-immobilized membrane with the electrode, the concentration of a substance such as a protein having a negative charge can be determined. The immune sensor can be used, for example, for diagnosing syphilis serum, reading blood types, and the like. In addition to the electrode type immune sensor, there is an immune sensor using a light measuring device, a crystal oscillator and the like.

On the other hand, in the conventional cancer diagnosis method, tumor markers specifically occurring in various cancers are detected from the blood. As a method for detecting tumor markers, an enzyme-linked immunosorbent assay (ELISA) is mainly used. However, as is known, such immunological assays are complex in their implementation and take a long time to obtain results.

Therefore, if a biosensor capable of selectively and quickly detecting tumor markers can be provided, the cancer diagnosis method can be significantly improved.

The present invention provides a biosensor for detecting a tumor marker, which can easily and selectively detect tumor markers in real time.

The present invention also provides a biosensor manufacturing method for detecting tumor markers.

The present invention also provides a method for analyzing tumor markers using the biosensor of the present invention described above.

Biosensor for detecting tumor markers provided by the present invention,

Board;

A first electrode attached to the substrate;

A second electrode attached to the substrate;

Semiconducting carbon nanotubes electrically connecting the first electrode and the second electrode;

A linker molecule whose one end is bonded to the surface of the semiconducting carbon nanotube; And

And bio-receptors for tumor markers, which are bound to the other end of the linker molecule.

Biosensor manufacturing method provided by the present invention,

(a) a substrate; A first electrode attached to the substrate; A second electrode attached to the substrate; Coupling a linker molecule to a transducer unit including a semiconducting carbon nanotube electrically connecting the first electrode and the second electrode; And

(b) binding a bio-receptor for a tumor marker to the linker molecule.

Tumor marker factor analysis method provided by the present invention,

Contacting the sample solution with the biosensor for detecting a tumor marker, as described above; And

And measuring a change in electrical conductivity of carbon nanotubes of the biosensor over time, while maintaining contact between the sample solution and the biosensor.

Hereinafter, the biosensor for detecting tumor markers of the present invention will be described in more detail. Tumor marker detection biosensor of the present invention is a substrate; A first electrode attached to the substrate; A second electrode attached to the substrate; Semiconducting carbon nanotubes electrically connecting the first electrode and the second electrode; A linker molecule whose one end is bonded to the surface of the semiconducting carbon nanotube; And a bio-receptor for a tumor marker that is bound to the other end of the linker molecule.

In the biosensor of the present invention, the first electrode, the second electrode, the semiconducting carbon nanotubes constitute a transducer portion, and the bioreceptor constitutes a sensor portion. The linker molecule serves to fix the bioreceptor on the surface of the semiconducting carbon nanotubes. One end of the linker molecule is bound to the surface of the semiconducting carbon nanotubes, and the other end of the linker molecule is bound to the bioreceptor. The bioreceptor is firmly fixed to the surface of the semiconducting carbon nanotubes by the linker molecule. The biosensor of the present invention constitutes a field effect transistor (FET) as a whole. The linker molecule acts as a gate insulator and the bioreceptor acts as a gate electrode.

The substrate may be any material having electrical insulation. For example, the substrate can be silicon, quartz, sapphire, glass, or plastic. For the growth of semiconducting carbon nanotubes, when using thermochemical vapor deposition, the substrate should be able to withstand high temperatures of about 900 ° C or higher. Therefore, the substrate is preferably silicon, quartz or sapphire. More preferably, silicon having a simple manufacturing process and relatively low cost can be used as the substrate.

The first electrode and the second electrode may be any material having electrical conductivity. For example, the first electrode and the second electrode may be gold, titanium, chromium, platinum, palladium, cobalt, or aluminum.

In the case of gold or aluminum, it tends to fall well from the silicon substrate. To prevent this phenomenon, titanium or chromium may be deposited on the substrate first, followed by gold or aluminum. Thus, the first electrode and the second electrode may be, for example, a multilayer electrode such as gold / titanium, gold / chromium, aluminum / titanium, or aluminum / chromium.

The first electrode may be used as a source electrode or a drain electrode. The second electrode may also be used as the source electrode or the drain electrode. However, if the first electrode is a source electrode, the second electrode is a drain electrode, and if the first electrode is a drain electrode, the second electrode is a source electrode.

The semiconducting carbon nanotubes electrically connect the first electrode and the second electrode. Semiconducting carbon nanotubes serve as charge transport channels.

The semiconducting carbon nanotubes may be p-type or n-type. Carbon nanotubes exhibiting semiconductor characteristics are typically p-type semiconductors in which charge carriers are holes. The n-type single-walled carbon nanotubes exhibiting n-type semiconductor characteristics in which the charge carriers are electrons can be obtained, for example, by doping K into the carbon nanotubes or by heat-treating the carbon nanotubes in vacuum.

The semiconducting carbon nanotubes may be single-walled, double-walled or multi-walled carbon nanotubes. In general, compared with double-walled or multi-walled carbon nanotubes, there is a greater possibility that single-walled carbon nanotubes have semiconductor characteristics. In addition, single-walled carbon nanotubes can be grown on a substrate very easily using a thermochemical vapor deposition method. Therefore, the semiconducting carbon nanotubes are preferably single walls.

The linker molecule serves to fix the bioreceptor close to the surface of the semiconducting carbon nanotubes. One end of the linker molecule is bound to the surface of the semiconducting carbon nanotube, and the other end of the linker molecule is bound to the bio-receptor.

The linker molecule has a first end (or a binding site) which can be bonded to the surface of the semiconducting carbon nanotubes through physical adsorption, chemical adsorption, covalent bonds, non-covalent bonds, and ionic bonds, and physical adsorption, chemical adsorption, and covalent bonds. Any nonconductive molecule having a second terminal (or binding site) capable of binding to the bio-receptor through binding, non-covalent and ionic bonding.

Typically, the surface of the carbon nanotubes are hydrophobic. Therefore, the first terminal of the linker molecule is preferably a hydrophobic moiety that can be easily bonded to the surface of the carbon nanotubes only by physical or chemical adsorption. For example, the first terminal may include a carbon chain having no substituent; Benzene ring; Pyrene; Or combinations thereof.

The second terminal of the linker molecule may be, for example, an imidazole group, which can easily form a chemical bond with the amine group when using an antibody having an amine group as a bio-receptor; Carboxyl groups; Or combinations thereof.

More specifically, for example, the linker molecule may be selected from the group consisting of CDI-twin20 (carbonyl diimidazol-tween20); 1-pyrenebutyric acid; Or combinations thereof. CDI-Twin 20 can be obtained by reacting CDI (carbonyl diimidazol) with Tween 20 (polyoxyethylene-20 sorbitan monolaurate) to bind CDI imidazole and Tween 20 hydroxy group.

Bio-receptors for tumor markers are bio-receptors having binding sites that can specifically bind tumor markers.

Tumor markers are bio-derived substances that can be used to diagnose cancer as a substance that specifically increases in vivo when cancer develops. Tumor indicators can be, for example, proteins, glycoproteins, or carbohydrates. Specific examples of tumor markers include AFP (alpha fetoprotein), CEA (carcinoembryonic antigen), CA (cancer antigen) 19-9, CA15-3, CA50, CA72-4, CA125, CA130, KMO-1, DuPAN-2, SPan-1, SLX, CA72-4, BFP, NCC-ST-439, SCC (sqamous cell carcinoma antigen), NSE (neuron specific enolase), CYFRA, γ-seminoprotein, prostate ACP, IAP, TPA (tissue polypeptide antigen) , Ferritin, PIVKA ii, polyamine, β-microglobulin, PSβ1, POA, PAP (prostatic acid phosphatase), Galectin-3, PSA (prostatic specific Ag), β2-MG (β2-microglobulin), and the like.

Tumor markers are mostly protein based skeletons, but may be glycoproteins or carbohydrates composed of a combination of protein and carbohydrate. Tumor markers that are glycoproteins are, for example, CEA. Examples of tumor markers that are carbohydrates include CA19-9.

Tumor marker bio-receptors are, for example, antibodies, enzymes, proteins, peptides, amino acids, nucleic acids, aptamers, lipids, noses having binding sites that can specifically bind to these tumor markers. It may be a factor or a carbohydrate.

Specific examples of the antibodies that can be used as bioreceptors for tumor markers include monoclonal antibodies, polyclonal antibodies, antibody binding fragments, and the like. .

In addition, inhibitors such as, for example, mycolic acid that specifically binds Galectin-3 can be used as bio-receptors for tumor markers.

Hereinafter, with reference to Figure 1, the structure and function of the biosensor of the present invention will be described in more detail. 1 is a cross-sectional view schematically showing one embodiment of the biosensor of the present invention.

The first electrode 200 and the second electrode 300 are provided on the substrate 100. The first electrode 200 and the second electrode 300 are electrically connected by the semiconducting carbon nanotubes 400. One end portion of the linker molecule 500 is bonded to the surface of the semiconducting carbon nanotubes 400. The other end portion of the linker molecule 500 is combined with a bio-receptor 600 for tumor markers.

For example, when detecting a carcinoembryonic antigen (CEA), which is a representative tumor marker of colorectal cancer, a monoclonal anti-CEA antibody may be used as the bio-receptor 600. CEA samples are prepared in the form of a buffer having a pH value of about 6.5. In buffers with a pH value of about 6.5, CEA is positively charged. Contacting the CEA sample with the biosensor of FIG. 1 specifically binds positively charged CEA to the bio-receptor 600, a monoclonal anti-CEA antibody. Thus, the bio-receptor 600 will have a positive potential proportional to the amount of CEA bound. If the semiconducting carbon nanotubes 400 have the characteristics of a p-type semiconductor, the electrical conductivity of the semiconducting carbon nanotubes 400 is reduced due to the electric field effect due to the positive potential formed in the bioreceptor 600. do. Accordingly, the current flowing along the semiconducting carbon nanotubes 400 is reduced while a predetermined voltage is applied between the first electrode 200 and the second electrode 300. By measuring this change in current, the presence and / or concentration of CEA in the CEA sample can be detected.

Since the bio-receptor 600 is fixed at the molecular level so as to be very close to the surface of the semiconducting carbon nanotubes 400, the electrical conductivity of the semiconducting carbon nanotubes 400 is determined by the bioreceptor 600. Dominated by the potential. In addition, since the bio-receptor 600 specifically binds to CEA without binding to other charging components in the CEA sample, the potential of the bio-receptor 600 undergoes a significant change only by the presence of CEA. As a result, the electrical conductivity of the semiconducting carbon nanotubes 400 may be changed only by CEA. Thus, when the monoclonal anti-CEA antibody is used as the bio-receptor 600, the biosensor of FIG. 1 can specifically detect only CEA.

As such, by using a bio-receptor that specifically binds each tumor marker, the biosensor of the present invention can specifically detect the tumor marker. In addition, since the potential change of the bio-receptor is made in real time by contact with the sample, the biosensor of the present invention is very fast (for example, within a few seconds to several minutes) and the presence of tumor markers in the sample and And / or the concentration thereof can be detected.

In another embodiment of the biosensor of the present invention, an insulating layer may be formed on at least one surface of the first electrode and the second electrode.

In the process of detecting the tumor marker in real time, the sample solution containing the tumor marker is dropped on the carbon nanotubes grown between the first electrode and the second electrode. At this time, the current must flow only through the carbon nanotube between the first electrode and the second electrode for accurate analysis. However, when the sample solution itself has electrical conductivity, current may flow between the first electrode and the second electrode through the sample solution. Therefore, the insulating layer is used to prevent current from flowing between the first electrode and the second electrode through the sample solution.

As the insulating layer, not only an insulating material commonly used in the field of a thin film transistor or an integrated device may be used, but a photoresist such as SU-8 may be used as the insulating material, for example. In particular, since SU-8 can exhibit very strong hydrophobicity after exposure to light, it can very effectively prevent the sample solution from contacting the electrode.

The present invention also provides a biosensor manufacturing method.

The biosensor manufacturing method of the present invention,

(a) a substrate; A first electrode attached to the substrate; A second electrode attached to the substrate; Coupling a linker molecule to a transducer unit including a semiconducting carbon nanotube electrically connecting the first electrode and the second electrode; And

(b) binding a bio-receptor for a tumor marker to the linker molecule.

The transducer portion used in step (a) may be obtained by, for example, horizontally growing carbon nanotubes on a substrate in the presence of a catalyst for growing carbon nanotubes and then forming an electrode pattern. Fabrication of the transducer element can be performed using a known carbon nanotube growth method and a known integrated circuit device fabrication method, and thus will not be described in detail in the present invention. The transducer unit may further include an insulating layer formed on at least one surface of the first electrode and the second electrode.

In step (a), the transducer unit element is brought into contact with the linker molecule to bond one end of the linker molecule to the surface of the semiconducting carbon nanotube. For example, by dipping the transducer element in a linker molecule-containing solution, the linker molecule and the semiconducting carbon nanotubes can be bonded.

Step (b) comprises contacting the transducer element and the tumor marker bio-receptor to which the linker molecule is bound so that the tumor marker bio is at the other end of the linker molecule bonded to the surface of the semiconducting carbon nanotube. -Joining the receptor. For example, by submerging the transducer element in which the linker molecule is bound in a solution containing the tumor-labeling bio-receptor, the bio-receptor for the tumor marker is attached to the other end of the linker molecule bound to the transducer. Can be combined.

By passing through the steps (a) and (b), a sensor unit, which is a combination of a linker molecule and a bio-receptor, is formed on the surface of the semiconducting carbon nanotube of the transducer unit.

The combination of linker molecule and bio-receptor may be bound in small amounts to other regions of the transducer portion other than the surface of the semiconducting carbon nanotubes. However, the combination of linker molecules and bio-receptors bound to the surface of semiconducting carbon or nanotubes dominates the field effect on semiconducting carbon nanotubes. Therefore, even if the linker molecule and the bio-receptor combination are present in other regions of the transducer part, there is no adverse effect on the function of the biosensor manufactured by the manufacturing method of the present invention.

In terms of improving the sensitivity of the biosensor, it is preferable that the conjugate of the linker molecule and the bio-receptor is concentrated to the tube of the semiconducting carbon nanotube. Thus, in another embodiment of the manufacturing method of the present invention, linker molecules that bind to the surface of the semiconducting carbon nanotubes better than the surface of the substrate and the electrode are used. For example, CDI-Tween20 (carbonyl diimidazol-tween20); Linker molecules, such as 1-pyrenebutyric acid, have superior binding preference to the surface of semiconducting carbon nanotubes.

By using the production method of the present invention, a biosensor for detecting tumor markers can be produced very easily.

The present invention also provides a method for analyzing tumor markers using the biosensor of the present invention described above.

Tumor marker factor analysis method of the present invention,

Contacting the sample solution with the biosensor for detecting a tumor marker, as described above; And

And measuring a change in electrical conductivity of carbon nanotubes of the biosensor over time, while maintaining contact between the sample solution and the biosensor.

The sample solution may contain a buffer solution for pH adjustment. As a solute solution for pH adjustment, for example, tris buffered saline (TBS), phosphate buffered saline (PBS), carbonate-bicarbonate, or a combination thereof may be used. As a solvent for the buffer solution for pH adjustment, deionized water can be used, for example.

As described above, using the tumor marker analysis method of the present invention, compared with conventional immunological techniques, it is possible to detect tumor markers with a very small amount of blood. This is because the sensitivity of the biosensor used in the method of the present invention is very excellent.

The pH value of the sample solution is preferably close to neutral because the biosensor is mainly based on the antigen-antibody reaction. For example, the pH of the sample solution may be about 6.4 to about 7.8.

The amount of the sample solution used to detect the tumor marker is not particularly limited, but usually, about 3 to about 5 μl. This amount is very small compared to the ELISA method which is a conventional immunological assay.

In the step of measuring the electrical conductivity change of the carbon nanotubes of the biosensor over time, if the time for maintaining the contact between the sample solution and the biosensor, or the time for measuring the electrical conductivity change is too small, accurate analysis is performed. Can't be done. In addition, since the analysis is made quickly, the time for measuring the change in conductivity does not have to be too long. In view of this, a preferred example of the conductivity change measurement time may be about 100 to about 600 seconds. As such, using the tumor marker analysis method of the present invention, it is possible to detect tumor markers very quickly compared to conventional immunological techniques.

From the rate of change of electrical conductivity over time obtained from the analysis method of the present invention, and / or the change of electrical conductivity over time, the presence and absence of a tumor marker in the sample and its concentration are very easy and very You can decide quickly.

Hereinafter, the biosensor of the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited to the following examples.

<Example>

Preparation Example 1 --- Fabrication of Transducer Parts

Semiconducting  Catalyst Solution Synthesis for Carbon Nanotube Growth

20 mg of iron (III) nitrate nonahydrate, 15 mg of aluminum oxide nanopowder and bis (acetylacetonato) dioxomolybdenum (VI) (bis (acetylacetonato) dioxomolybdenum (VI) 5 mg was added to 15 ml of methanol and stirred at room temperature for 24 hours to obtain a catalyst solution.

Formation of catalyst pattern

After coating PMMA (polymethyl methacrylate) on the silicon substrate by spin coating, a catalyst site pattern was formed by electron beam lithography. After stirring the catalyst solution for 24 hours by sonication for 1 hour, the catalyst solution was dropped on the silicon substrate on which the catalyst site pattern was formed for 20 seconds, and then the excess catalyst solution was removed using an air gun. This substrate was dried in a 150 ° C. oven for 3 minutes. The substrate catalyzed by acetone at 50 ° C. was immersed for 30 seconds to remove PMMA from the surface of the substrate, and then the substrate was washed with acetone and isopropyl alcohol.

Semiconducting  Growth of Carbon Nanotubes

The silicon substrate on which the catalyst pattern was formed was placed in a quartz tube in a furnace and heated to 900 ° C. while flowing Ar of 1000 SCCM. When the furnace temperature reached 900 ° C., the Ar feed was stopped and then 5000 SCCM of methane and 600 SCCM of hydrogen were fed for 12 minutes. Then, the supply of methane and hydrogen was stopped and the furnace was cooled to room temperature while supplying Ar of 1000 SCCM. Through this process, a silicon substrate on which semiconducting single-walled carbon nanotubes were formed between catalyst patterns was obtained.

Formation of electrodes

After the photoresist (AZ7210) was coated on the silicon substrate on which the semiconductor carbon nanotubes were grown by spin coating, an electrode pattern was formed by photolithography, and then, by using a thermal evaporator. An nm thick titanium layer and a 20 nm gold layer were deposited. The device was immersed in acetone and lifted off.

SU -8 Insulation layer  formation

After SU-8 negative photoresist was applied on the device on which the electrode was formed by spin coating, an insulating layer pattern was formed by photolithography.

Through this process, a transducer unit device including a silicon substrate, a first electrode, a second electrode, and semiconducting carbon nanotubes was manufactured. Fig. 2 is an AFM (atomic force microscope) photograph of the transducer part element manufactured in the present example. As shown in FIG. 2, horizontally grown semiconducting single-walled carbon nanotubes electrically connect both electrodes.

Production Example 2-Synthesis of CDI-Tween20

In this example, CDI-Tween20 to be used as a linker molecule was synthesized. In a 10 ml glass container, 600 mg of CDI, 5 ml of diethyl ether and 1 ml of Tween20 were sequentially added. The reaction was carried out at room temperature for 2 hours while the inlet of the glass container was opened. The lower solution of the phase separated reaction solution was transferred to a new 10 ml glass container using a pipette. After diethyl ether was added to the glass container and mixed well, the synthesized CDI-Tween20 and diethyl ether were allowed to phase separate, and then the upper diethyl ether was removed using a pipette. The last procedure was repeated five times, and finally, the separated CDI-Tween20 was dried at room temperature for 2 hours using a vacuum apparatus to remove the remaining diethyl ether.

Example 1 Manufacture of Biosensor

The transducer unit device obtained in Preparation Example 1 was immersed for 2 hours in a CDI-Tween20 solution (CDI-Tween20: deionized water = 5: 1130 (v / v)) obtained in Preparation Example 2, thereby being a linker molecule. CDI-Tween20 was combined with the transducer element.

Subsequently, the transducer element was immersed in a 10 mM PBS solution containing monoclonal anti-CEA antibody (Sigma-Aldrich) at a concentration of 18 ng / ml IgG, and then reacted for 5 hours while maintaining the temperature at 20 ° C. By doing so, CDI-Tween20 and monoclonal anti-CEA antibody were bound.

Thus, the biosensor of Example 1 was produced. 3 is an AFM photograph showing that the bioreceptor is immobilized on the surface of the carbon nanotube of the biosensor of Example 1. FIG. In FIG. 3, the bright yellow dots are monoclonal anti-CEA antibodies bound to CDI-Tween20. It can be seen that these bright yellow dots are concentrated along the surface of the carbon nanotubes (the dark yellow dots on the silicon substrate are not monoclonal anti-CEA antibodies and are not treated with AFM images of the silicon substrate). Is also a common point).

Example 2 Performance of Biosensor

In this example, the CEA detection capability of the biosensor obtained in Example 1 was confirmed.

First, 1683 μl of 1 mM PBS buffer solution was added to 17 μl of Sigma-Aldrich's CEA solution (1.6 mg / ml) to obtain a CEA solution having a concentration of 16 μg / ml. The CEA solution was then diluted again with 1 mM PBS buffer solution, CEA sample solution at 5.4 μg / ml, CEA sample solution at 5.4 ng / ml, CEA sample solution at 5.4 pg / ml and 5.4 pg / ml A concentration of CEA sample solution was obtained.

Then, a circuit for voltage application and current measurement was connected to two electrodes of the biosensor obtained in Example 1. The voltage applied between the two electrodes of the biosensor was 100 mV. "Probe Station" and "LabVIEW ^TM v6.1" were used as instruments for measuring the current through the carbon nanotubes.

Then, 5 μl of each of the four CEA sample solutions was sequentially dropped on the surface of the biosensor, and then, as time passed, the change in the current passing through the carbon nanotubes was measured. Results for four CEA samples are shown in FIG. 4. 4 is a graph showing current changes of four CEA samples measured in Example 2. FIG.

As shown in FIG. 4, each time four CEA sample solutions were added to the biosensor of Example 1, the current passing through the carbon nanotubes decreased. Therefore, the biosensor of Example 1 shows the performance of the p-type FET, and the positive field effect as the CEA binds to the bioreceptor (monoclonal anti-CEA antibody) fixed on the surface of the carbon nanotubes. The electrical conductivity of the carbon nanotubes decreased, and as the CEA concentration increased, the electrical conductivity of the carbon nanotubes decreased step by step. In addition, it can be seen from the results of FIG. 4 that the CEA-detecting biosensor of Example 1 exhibits sensitivity even at very low concentrations of CEA sample solutions ranging from several pg to several ng / ml.

Example 3 --- Performance of Biosensor

In the present Example, by the same method as Example 2, after preparing the CEA sample solution of 40 ng / ㎖ concentration, the CEA sample solution of 0.4 ㎍ / ㎖ concentration and CEA sample solution of 40 ㎍ / ㎖ concentration, these three Using the CEA sample solution, the CEA detection capability of the biosensor obtained in Example 1 was confirmed. Results for three CEA sample solutions are shown in FIG. 5. FIG. 5 is a graph showing current changes of three CEA samples measured in Example 3. FIG.

As shown in FIG. 5, each time three CEA sample solutions were added to the biosensor of Example 1, the current passing through the carbon nanotubes was gradually decreased. The current reduction value when dropping the CEA sample solution at the concentration of 40 ng / ㎖ was ΔI ₁ , the current reduction value when dropping 0.4A / CEA sample solution was ΔI ₂ , CEA sample at 40 ㎍ / ㎖ concentration The current reduction value when dropping the solution was ΔI ₃ .

6 shows the correlation between the ratio (%) of the current reduction value (ΔI) to the initial current (I ₀ ) with no CEA sample solution dropping and the CEA concentration using the results shown in FIG. 5. The graph shown. As shown in Figure 6, it can be seen that the current decrease value increases with increasing CEA concentration. By using this correlation, the concentration of the tumor marker in the sample solution can be measured very easily using the biosensor of the present invention. Although the correlation of FIG. 6 is shown by a straight line, other forms of correlation may be obtained depending on specific measurement conditions.

Biosensors for tumor markers of the present invention include very good selective specificity for tumor markers; And very good sensitivity capable of detecting very low concentrations of tumor markers.

By using the manufacturing method of the present invention, a biosensor for detecting tumor markers can be produced very easily.

By using the tumor marker analysis method of the present invention, compared to conventional immunological techniques, it is possible to detect tumor markers with a very small amount of blood. In addition, using the tumor marker analysis method of the present invention, it is possible to detect tumor markers very quickly compared to conventional immunological techniques.

Claims (18)

Board; A first electrode attached to the substrate; A second electrode attached to the substrate; Semiconducting carbon nanotubes electrically connecting the first electrode and the second electrode; A linker molecule whose one end is bonded to the surface of the semiconducting carbon nanotube; And It includes; bio-receptor for tumor markers that are bound to the other end of the linker molecule, and Wherein the linker molecule is CDI-Twin20, 1-pyrenbutyric acid, or a combination thereof, Biosensor for detecting tumor markers. The biosensor for detecting a tumor marker according to claim 1, wherein the semiconducting carbon nanotubes are single walls. delete delete delete The method of claim 1, wherein the bioreceptor for tumor markers is AFP (alpha fetoprotein), CEA (carcinoembryonic antigen), CA (cancer antigen) 19-9, CA15-3, CA50, CA72-4, CA125, CA130 , KMO-1, DuPAN-2, SPan-1, SLX, CA72-4, BFP, NCC-ST-439, SCC (sqamous cell carcinoma antigen), NSE (neuron specific enolase), CYFRA, γ-seminoprotein, prostate ACP , IAP, tissue polypeptide antigen (TPA), Ferritin, PIVKA ii, polyamine, β-microglobulin, PSβ1, POA, PAP (prostatic acid phosphatase), Galectin-3, PSA (prostatic specific Ag) or β2-MG (β2-microglobulin A biosensor for detecting a tumor marker, characterized in that the bio-receptor having a binding site that can specifically bind to). The biosensor for detecting a tumor marker according to claim 1, wherein the tumor marker bio-receptor is an antibody, enzyme, protein, peptide, amino acid, nucleic acid, aptamer, lipid, cofactor or carbohydrate. The biosensor for detecting a tumor marker according to claim 1, wherein the tumor marker bio-receptor is a monoclonal antibody, a polyclonal antibody or an antibody binding site fragment. The biosensor for tumor marker detection according to claim 1, wherein the tumor marker bio-receptor is an inhibitor. The biosensor for detecting a tumor marker according to claim 1, wherein an insulating layer is formed on at least one of the first electrode and the second electrode. delete (a) a substrate; A first electrode attached to the substrate; A second electrode attached to the substrate; Coupling a linker molecule to a transducer unit including a semiconducting carbon nanotube electrically connecting the first electrode and the second electrode; And (b) binding a bio-receptor for a tumor marker to the linker molecule; The linker molecule is CDI-Tween20; 1-pyrenbutyric acid; Or a combination thereof, Biosensor manufacturing method. The biosensor manufacturing method according to claim 12, wherein the transducer unit further comprises an insulating layer formed on at least one surface of the first electrode and the second electrode. delete Contacting the sample solution with a biosensor for detecting a tumor marker according to any one of claims 1, 2 and 6 to 10; And And measuring a change in electrical conductivity of carbon nanotubes of the biosensor over time, while maintaining contact between the sample solution and the biosensor. 16. The method of claim 15, wherein the sample solution contains a buffer solution for pH adjustment. 16. The method of claim 15, wherein the pH value of the sample solution is 6.4 to 7.8. The method of claim 15, wherein the time for maintaining contact between the sample solution and the biosensor is 100 to 600 seconds.

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